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1.  CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma 
British Journal of Cancer  2013;110(4):958-966.
Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC).
Circulating CD44+CD90+ cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro.
CD44+CD90+ cells in the blood acquired epithelial–mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44high population showed higher anoikis resistance and sphere-forming ability than did the CD44low population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44high population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown.
CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.
PMCID: PMC3929866  PMID: 24300972
CD44; epithelial–mesenchymal transition; Twist1; anoikis
2.  Particle therapy using carbon ions or protons as a definitive therapy for patients with primary sacral chordoma 
The British Journal of Radiology  2013;87(1033):20130512.
This study retrospectively evaluated the efficacy and toxicity of particle therapy using carbon ions or protons for primary sacral chordomas.
We evaluated 23 patients with primary sacral chordoma treated with carbon ion therapy (CIT) or proton therapy (PT) between July 2005 and June 2011 at the Hyogo Ion Beam Medical Center, Hyogo, Japan. The median patient age was 72 years. 14 patients were treated with 70.4 Gy equivalents (GyE) in 16 fractions and 9 were treated with 70.4 GyE in 32 fractions. CIT was used for 16 patients, and PT was used for 7 patients.
The median follow-up period was 38 months. At 3 years, local control (LC), overall survival (OS) and progression-free survival (PFS) for all patients were 94%, 83% and 68%, respectively. The log-rank test revealed that male sex was significantly related to better PFS ( p = 0.029). No other factors, including dose fractionation and ion type, were significant for LC, OS or PFS. In nine patients, ≥Grade 3 acute dermatitis was observed, and ≥Grade 3 late toxicities were observed in nine patients. The 32-fraction protocol reduced severe toxicities in both the acute and late phases compared with the 16-fraction protocol.
Particle therapy for patients with sacral chordoma showed favourable LC and OS. Severe toxicities were successfully reduced by modifying the dose fractionation and treatment planning in the later treatment era. Thus, this therapeutic modality should be considered useful and safe.
Advances in knowledge:
This is the first study including both CIT and PT for sacral chordomas.
PMCID: PMC3898974  PMID: 24288399
3.  Acceleration of periosteal bone formation by human basic fibroblast growth factor containing a collagen-binding domain from Clostridium histolyticum collagenase 
Basic fibroblast growth factor 2 (bFGF) is a potent mitogen for mesenchymal cells, and the local application of recombinant bFGF accelerates bone union and defect repair. However, repeated dosing is required for sustained therapeutic effect as the efficacy of bFGF decreases rapidly following its diffusion from bone defect sites. Here, we attempted to develop a collagen-based bone formation system using a fusion protein (collagen binding-bFGF, CB-bFGF) consisting of bFGF and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The addition of the CBD to bFGF did not modify its native biological activity, as shown by the capacity of the fusion protein to promote the in vitro proliferation of periosteal mesenchymal cells. The affinity of the fusion protein towards collagen and demineralized bone matrix (DBM) was also confirmed by collagen-binding assays. Moreover, in vivo periosteal bone formation assays showed that the combination of CB-bFGF with a collagen sheet induced periosteal bone formation at protein concentrations lower than those required for bFGF alone. In addition, grafts of DBM loaded with CB-bFGF accelerated new bone formation in rat femurs compared to the same concentration of bFGF administered alone. Taken together, these properties suggest that the CB-bFGF/collagen composite is a promising material for bone repair in the clinical setting. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1737–1743, 2014.
PMCID: PMC4232007  PMID: 23775724
basic fibroblast growth factor; bone tissue engineering; bone repair; collagen; collagen-binding domain
4.  Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro 
Biology Open  2014;3(11):1108-1115.
Rab GTPases are master regulators of eukaryotic endomembrane systems, particularly functioning in membrane tethering to confer the directionality of intracellular membrane trafficking. However, how exactly Rab GTPases themselves act upon membrane tethering processes has remained enigmatic. Here, we thoroughly tested seven purified Rab GTPases in human, which localize at the various representative organelles, for their capacity to support membrane tethering in vitro. Strikingly, we found that three specific human Rabs (endoplasmic reticulum/Golgi Rab2a, early endosomal Rab5a, and late endosomal/lysosomal Rab7a) strongly accelerated membrane aggregation of synthetic liposomes even in the absence of any additional components, such as classical tethers, tethering factors, and Rab effectors. This Rab-induced membrane aggregation was a reversible membrane tethering reaction that can be strictly controlled by the membrane recruitment of Rab proteins on both apposing membranes. Thus, our current reconstitution studies establish that membrane-anchored human Rab GTPases are an essential tethering factor to directly mediate membrane tethering events.
PMCID: PMC4232769  PMID: 25361579
Rab GTPase; Liposome; Membrane tethering; Membrane traffic; Reconstitution
6.  Tumor Compression–Induced Portal Obstruction and Selective Transarterial Chemoembolization Increase Functional Liver Volume in the Unobstructed Area, Facilitating Successful Resection of a Large HCC 
International Surgery  2013;98(4):388-391.
A 62-year-old man with hepatitis B was admitted for treatment of a large hepatocellular carcinoma. The right portal vein was completely obstructed by tumor compression. Although we initially planned a right trisectionectomy as curative hepatectomy, the percentage of future remnant liver volume (%RLV) and the percentage of functional liver volume (%RFLV) were 31.2% and 41.3%, respectively. Because %RFLV showed marginal tolerability for curative hepatectomy and %RLV was very low, we opted for transarterial chemoembolization of segment IV and the right lobe containing the tumor as an approach to selectively reduce liver volume and abolish liver function. One month later, %RLV and %RFLV had dramatically increased to 46.6% and 67.2%, resulting in curative hepatectomy. Our results suggest that tumor compression–induced portal obstruction and selective transarterial chemoembolization increase %RFLV much more than %RLV. This may represent a useful approach in preoperative management in patients with large hepatocellular carcinomas to improve %RFLV for hepatic resection.
PMCID: PMC3829069  PMID: 24229029
Hepatocellular carcinoma; Portal vein obstruction; Transarterial chemoembolization; Functional liver volume
7.  Induction of Vascular Insulin Resistance, Endothelin-1 Expression and Acceleration of Atherosclerosis by the Overexpression of Protein Kinase C β Isoform in the Endothelium 
Circulation research  2013;113(4):418-427.
Loss of insulin action on the endothelium can cause endothelial dysfunction and atherosclerosis. Hyperglycemia and elevated fatty acids induced by diabetes can activate protein kinase C (PKC) β isoforms and selectively inhibit insulin signaling via phosphatidylinositol 3-kinase (PI3K)/Akt pathway to inhibit the activation of endothelial nitric oxide synthase (eNOS) and metabolic actions.
To demonstrate that overexpressing PKCβ2 isoform in endothelial cells can cause selective insulin resistance and exacerbate atherosclerosis in the aorta.
Methods and Results
PKCβ2 isoform was overexpressed in endothelial cells using a promoter of vascular endothelial cell-cadherin (VE-Cadherin). These mice were cross-bred with ApoE-/- mice (Tg (Prkcb)ApoE-/-). On a Western diet, Tg(Prkcb)ApoE-/- and ApoE-/- mice did not differ in systemic insulin sensitivity, glucose tolerance, plasma lipid or blood pressure. Insulin action in endothelial cells and femoral artery from Tg(Prkcb)ApoE-/- mice were impaired by ∼40% with respect to Akt/eNOS activation and leukocyte-endothelial cell binding increased in cultured lung endothelial cells from Tg(Prkcb)ApoE-/-mice compared to ApoE-/- mice. Basal and angiotensin stimulated big endothelin-1 (ET-1) levels were elevated in Tg(Prkcb)ApoE-/- mice compared to ApoE-/- mice. The severity of atherosclerosis in the aorta from Tg(Prkcb)ApoE-/- mice increased by ∼70% as measured by en face fat staining and plaque content of the number of smooth muscle cells, macrophages and extracellular matrix.
Specific PKCβ2 activation in the endothelial cells caused dysfunction and accelerated atherosclerosis due to loss of insulin-stimulated Akt/eNOS activation and angiotensin induced increases in ET-1 expression.
PMCID: PMC3893704  PMID: 23759514
Endothelium; endothelial dysfunction; protein kinase C; insulin resistance; atherosclerosis
8.  ‘Putting Life in Years’ (PLINY) telephone friendship groups research study: pilot randomised controlled trial 
Trials  2014;15:141.
Loneliness in older people is associated with poor health-related quality of life (HRQoL). We undertook a parallel-group randomised controlled trial to evaluate the effectiveness and cost-effectiveness of telephone befriending for the maintenance of HRQoL in older people. An internal pilot tested the feasibility of the trial and intervention.
Participants aged >74 years, with good cognitive function, living independently in one UK city were recruited through general practices and other sources, then randomised to: (1) 6 weeks of short one-to-one telephone calls, followed by 12 weeks of group telephone calls with up to six participants, led by a trained volunteer facilitator; or (2) a control group. The main trial required the recruitment of 248 participants in a 1-year accrual window, of whom 124 were to receive telephone befriending. The pilot specified three success criteria which had to be met in order to progress the main trial to completion: recruitment of 68 participants in 95 days; retention of 80% participants at 6 months; successful delivery of telephone befriending by local franchise of national charity. The primary clinical outcome was the Short Form (36) Health Instrument (SF-36) Mental Health (MH) dimension score collected by telephone 6 months following randomisation.
We informed 9,579 older people about the study. Seventy consenting participants were randomised to the pilot in 95 days, with 56 (80%) providing valid primary outcome data (26 intervention, 30 control). Twenty-four participants randomly allocated to the research arm actually received telephone befriending due to poor recruitment and retention of volunteer facilitators. The trial was closed early as a result. The mean 6-month SF-36 MH scores were 78 (SD 18) and 71 (SD 21) for the intervention and control groups, respectively (mean difference, 7; 95% CI, -3 to 16).
Recruitment and retention of participants to a definitive trial with a recruitment window of 1 year is feasible. For the voluntary sector to recruit sufficient volunteers to match demand for telephone befriending created by trial recruitment would require the study to be run in more than one major population centre, and/or involve dedicated management of volunteers.
Trial registration
PMCID: PMC4022155  PMID: 24758530
9.  The MAP Kinase Slt2 Is Involved in Vacuolar Function and Actin Remodeling in Saccharomyces cerevisiae Mutants Affected by Endogenous Oxidative Stress 
Applied and Environmental Microbiology  2013;79(20):6459-6471.
Oxidative stress causes transient actin cytoskeleton depolarization and also provokes vacuole fragmentation in wild-type cells. Under conditions of oxidative stress induced by hydrogen peroxide, the Slt2 protein is required to repolarize the actin cytoskeleton and to promote vacuole fusion. In this study, we show that grx3 grx4 and grx5 mutants are cellular models of endogenous oxidative stress. This stress is the result of alterations in iron homeostasis that lead to impairment of vacuolar function and also to disorganization of the actin cytoskeleton. Slt2 overexpression suppresses defects in vacuolar function and actin cytoskeleton organization in the grx3 grx4 mutant. Slt2 exerts this effect independently of the intracellular levels of reactive oxygen species (ROS) and of iron homeostasis. The deletion of SLT2 in the grx3 grx4 mutant results in synthetic lethality related to vacuolar function with substantial vacuole fragmentation. The observation that both Vps4 and Vps73 (two proteins related to vacuole sorting) suppress vacuole fragmentation and actin depolarization in the grx3 grx4 slt2 triple mutant strengthens the hypothesis that Slt2 plays a role in vacuole homeostasis related to actin dynamics. Here we show that in sod1, grx5, and grx3 grx4 slt2 mutants, all of which are affected by chronic oxidative stress, the overexpression of Slt2 favors vacuole fusion through a mechanism dependent on an active actin cytoskeleton.
PMCID: PMC3811184  PMID: 23956390
10.  Combat Diabetic Nephropathy: From Pathogenesis to Treatment 
Journal of Diabetes Research  2014;2014:207140.
PMCID: PMC3972827  PMID: 24741570
11.  Multiple and distinct strategies of yeast SNAREs to confer the specificity of membrane fusion 
Scientific Reports  2014;4:4277.
Trans-QabcR-SNARE pairing on opposing membranes is crucial for eukaryotic membrane fusion, but how selective pairs of Qabc- and R-SNARE proteins regulate membrane fusion specificity remains elusive. Here, we studied 14 purified full-length SNAREs that function in yeast endoplasmic reticulum (ER)-Golgi, intra-Golgi, endosomal, and vacuolar transport by comprehensively testing cis-QabcR-SNARE assembly and fusogenicity of reconstituted SNARE proteoliposomes. Strikingly, the cognate ER-Golgi and intra-Golgi SNARE-complex assemblies were highly stringent, whereas endosomal and vacuolar SNAREs assembled rather promiscuously into the non-cognate mixed complexes. However, these patterns of cis-SNARE assemblies cannot solely explain their potency to be fusogenic via trans-SNARE pairing: Only the vacuolar 3Q-SNARE combination is fusogenic in the absence of additional components; endosomal SNARE-dependent fusogenicity requires membrane-tethering factors; and ER-Golgi SNAREs can be fusogenic by synergistic actions of tethering factors and the cognate Sec1/Munc18-family protein Sly1p. Thus, our findings uncover multiple and distinct strategies of SNAREs to directly mediate fusion specificity.
PMCID: PMC3940976  PMID: 24589832
12.  Receptor for Advanced Glycation End Products Regulates Adipocyte Hypertrophy and Insulin Sensitivity in Mice 
Diabetes  2013;62(2):478-489.
Receptor for advanced glycation end products (RAGE) has been shown to be involved in adiposity as well as atherosclerosis even in nondiabetic conditions. In this study, we examined mechanisms underlying how RAGE regulates adiposity and insulin sensitivity. RAGE overexpression in 3T3-L1 preadipocytes using adenoviral gene transfer accelerated adipocyte hypertrophy, whereas inhibitions of RAGE by small interfering RNA significantly decrease adipocyte hypertrophy. Furthermore, double knockdown of high mobility group box-1 and S100b, both of which are RAGE ligands endogenously expressed in 3T3-L1 cells, also canceled RAGE-medicated adipocyte hypertrophy, implicating a fundamental role of ligands–RAGE ligation. Adipocyte hypertrophy induced by RAGE overexpression is associated with suppression of glucose transporter type 4 and adiponectin mRNA expression, attenuated insulin-stimulated glucose uptake, and insulin-stimulated signaling. Toll-like receptor (Tlr)2 mRNA, but not Tlr4 mRNA, is rapidly upregulated by RAGE overexpression, and inhibition of Tlr2 almost completely abrogates RAGE-mediated adipocyte hypertrophy. Finally, RAGE−/− mice exhibited significantly less body weight, epididymal fat weight, epididymal adipocyte size, higher serum adiponectin levels, and higher insulin sensitivity than wild-type mice. RAGE deficiency is associated with early suppression of Tlr2 mRNA expression in adipose tissues. Thus, RAGE appears to be involved in mouse adipocyte hypertrophy and insulin sensitivity, whereas Tlr2 regulation may partly play a role.
PMCID: PMC3554382  PMID: 23011593
13.  Serine Phosphorylation Sites on IRS2 Activated by Angiotensin II and Protein Kinase C To Induce Selective Insulin Resistance in Endothelial Cells 
Molecular and Cellular Biology  2013;33(16):3227-3241.
Protein kinase C (PKC) activation, induced by hyperglycemia and angiotensin II (AngII), inhibited insulin-induced phosphorylation of Akt/endothelial nitric oxide (eNOS) by decreasing tyrosine phosphorylation of IRS2 (p-Tyr-IRS2) in endothelial cells. PKC activation by phorbol ester (phorbol myristate acetate [PMA]) reduced insulin-induced p-Tyr-IRS2 by 46% ± 13% and, similarly, phosphorylation of Akt/eNOS. Site-specific mutational analysis showed that PMA increased serine phosphorylation at three sites on IRS2 (positions 303, 343, and 675), which affected insulin-induced tyrosine phosphorylation of IRS2 at positions 653, 671, and 911 (p-Tyr-IRS2) and p-Akt/eNOS. Specific PKCβ2 activation decreased p-Tyr-IRS2 and increased the phosphorylation of two serines (Ser303 and Ser675) on IRS2 that were confirmed in cells overexpressing single point mutants of IRS2 (S303A or S675A) containing a PKCβ2-dominant negative or selective PKCβ inhibitor. AngII induced phosphorylation only on Ser303 of IRS2 and inhibited insulin-induced p-Tyr911 of IRS2 and p-Akt/eNOS, which were blocked by an antagonist of AngII receptor I, losartan, or overexpression of single mutant S303A of IRS2. Increases in p-Ser303 and p-Ser675 and decreases in p-Tyr911 of IRS2 were observed in vessels of insulin-resistant Zucker fatty rats versus lean rats. Thus, AngII or PKCβ activation can phosphorylate Ser303 and Ser675 in IRS2 to inhibit insulin-induced p-Tyr911 and its anti-atherogenic actions (p-Akt/eNOS) in endothelial cells.
PMCID: PMC3753901  PMID: 23775122
14.  The anodal tDCS over the left posterior parietal cortex enhances attention toward a focus word in a sentence 
The posterior parietal cortex (PPC) has two attentional functions: top-down attentional control and stimulus-driven attentional processing. Using the focused version of the reading span test (RST), in which the target word to be remembered is the critical word for comprehending a sentence (focused word) or a non-focused word, we examined the effect of tDCS on resolution of distractor interference by the focused word in the non-focus condition (top-down attentional control) and on augmented/shrunk attentional capture by the focused word in both the focus and non-focus conditions (stimulus-driven attentional processing). Participants were divided into two groups: anodal tDCS (atDCS) and cathodal tDCS (ctDCS). Online stimulation was given while participants performed the RST. A post-hoc recognition task was also administered in which three kinds of words were presented: target words in the RST, distractor words in the RST, and novel words. atDCS augmented the effect of the focused word by increasing differences in performance between the focus and non-focus conditions. Such an effect was not observed in the ctDCS group. As for the recognition task, atDCS again produced the augmented effect of the focused words in the distractor recognition. On the other hand, ctDCS brought less recognition of non-focused target words in comparison to sham. The results indicate that atDCS promotes stimulus-driven attentional processing, possibly by affecting neural firing in the inferior parietal regions. In contrast, ctDCS appears to prevent retrieval of less important information from episodic memory, which may require top-down attentional processing.
PMCID: PMC4260498  PMID: 25538609
posterior parietal cortex; tDCS; attention; working memory; reading span test
15.  Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses 
PLoS ONE  2013;8(11):e80368.
Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4+ T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4+ T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster.
PMCID: PMC3832426  PMID: 24260378
16.  Protective Effects of GLP-1 on Glomerular Endothelium and Its Inhibition by PKCβ Activation in Diabetes 
Diabetes  2012;61(11):2967-2979.
To characterize glucagon-like peptide (GLP)-1 signaling and its effect on renal endothelial dysfunction and glomerulopathy. We studied the expression and signaling of GLP-1 receptor (GLP-1R) on glomerular endothelial cells and the novel finding of protein kinase A–dependent phosphorylation of c-Raf at Ser259 and its inhibition of angiotensin II (Ang II) phospho–c-Raf(Ser338) and Erk1/2 phosphorylation. Mice overexpressing protein kinase C (PKC)β2 in endothelial cells (EC-PKCβ2Tg) were established. Ang II and GLP-1 actions in glomerular endothelial cells were analyzed with small interfering RNA of GLP-1R. PKCβ isoform activation induced by diabetes decreased GLP-1R expression and protective action on the renal endothelium by increasing its degradation via ubiquitination and enhancing phospho–c-Raf(Ser338) and Ang II activation of phospho-Erk1/2. EC-PKCβ2Tg mice exhibited decreased GLP-1R expression and increased phospho–c-Raf(Ser338), leading to enhanced effects of Ang II. Diabetic EC-PKCβ2Tg mice exhibited greater loss of endothelial GLP-1R expression and exendin-4–protective actions and exhibited more albuminuria and mesangial expansion than diabetic controls. These results showed that the renal protective effects of GLP-1 were mediated via the inhibition of Ang II actions on cRaf(Ser259) and diminished by diabetes because of PKCβ activation and the increased degradation of GLP-1R in the glomerular endothelial cells.
PMCID: PMC3478518  PMID: 22826029
17.  Implications of Treatment That Target Protective Mechanisms Against Diabetic Nephropathy 
Seminars in nephrology  2012;32(5):471-478.
Diabetes results in vascular changes and dysfunction, and vascular complications are the leading cause of morbidity and mortality in diabetic patients. There has been a continual increase in the number of diabetic nephropathy patients and epidemic increases in the number of patients progressing to end-stage renal diseases. To identify targets for therapeutic intervention, most studies have focused on understanding how abnormal levels of glucose metabolites cause diabetic nephropathy, which is of paramount importance in devising strategies to combat the development and progression of diabetic nephropathy. However, less studied than the systemic toxic mechanisms, hyperglycemia and dyslipidemia might inhibit the endogenous vascular protective factors such as insulin, vascular endothelial growth factor, and platelet-derived growth factor. In this review, we highlight the importance of enhancing endogenous protective factors to prevent or delay diabetic nephropathy.
PMCID: PMC3584697  PMID: 23062988
Hyperglycemia; protein kinase C (PKC)β; advanced glycation end products (AGEs); platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF)
18.  Dual Involvement of Growth Arrest-Specific Gene 6 in the Early Phase of Human IgA Nephropathy 
PLoS ONE  2013;8(6):e66759.
Gas6 is a growth factor that causes proliferation of mesangial cells in the development of glomerulonephritis. Gas6 can bind to three kinds of receptors; Axl, Dtk, and Mer. However, their expression and functions are not entirely clear in the different glomerular cell types. Meanwhile, representative cell cycle regulatory protein p27 has been reported to be expressed in podocytes in normal glomeruli with decreased expression in proliferating glomeruli, which inversely correlated with mesangial proliferation in human IgA nephropathy (IgAN).
The aim of this study is to clarify Gas6 involvement in the progression of IgAN. Expression of Gas6/Axl/Dtk was examined in 31 biopsy proven IgAN cases. We compared the expression levels with histological severity or clinical data. Moreover, we investigated the expression of Gas6 and its receptors in cultured podocytes.
In 28 of 31 cases, Gas6 was upregulated mainly in podocytes. In the other 3 cases, Gas6 expression was induced in endothelial and mesangial cells, which was similar to animal nephritis models. Among 28 podocyte type cases, the expression level of Gas6 correlated with the mesangial hypercellularity score of IgAN Oxford classification and urine protein excretion. It also inversely correlated with p27 expression in glomeruli. As for the receptors, Axl was mainly expressed in endothelial and mesangial cells, while Dtk was expressed in podocytes. In vitro, Dtk was expressed in cultured murine podocytes, and the expression of p27 was decreased by Gas6 stimulation.
Gas6 was uniquely upregulated in either endothelial/mesangial cells or podocytes in IgAN. The expression pattern can be used as a marker to classify IgAN. Gas6 has a possibility to be involved in not only mesangial proliferation via Axl, but also podocyte injury via Dtk in IgAN.
PMCID: PMC3691258  PMID: 23826128
19.  Gene expression of bone morphogenic protein 8B in the primary site, peripheral blood and bone marrow of patients with gastric cancer 
Oncology Letters  2013;6(2):387-392.
The prognosis for individuals that are diagnosed with gastric cancer remains poor due to the high frequency of metastatic disease. In response to tumor-derived secreted factors, the bone marrow generates a suitable microenvironment for the development of metastasis. However, it is largely unknown whether secreted factors in bone marrow associated with metastatic disease of patients with gastric cancer are present. Secreted factors from the bone marrow of patients with metastatic gastric cancer were identified using a DNA microarray analysis and the mRNA expression levels were investigated in 355 bone marrow, 295 peripheral blood and 144 primary site samples using quantitative PCR (qPCR). Using DNA microarray analysis, the present study identified bone morphogenetic protein 8B (BMP8B) as a secreted signaling molecule in the bone marrow that was associated with the metastatic disease of human gastric cancer. The expression levels of BMP8B in the bone marrow of 355 gastric cancer patients were increased with metastatic disease. A significant correlation was demonstrated between BMP8B mRNA expression in the bone marrow and in the peripheral blood. High BMP8B expression in the bone marrow was associated with the diffuse type of gastric cancer (P=0.009), lymph node metastasis (P=0.009), liver metastasis (P=0.044) and peritoneal dissemination (P<0.001). In the primary site, a multivariate analysis revealed BMP8B mRNA expression as one of the independent prognostic factors of gastric cancer [hazard ratio (HR), 2.066; 95% CI, 1.132–3.772]. This study suggests that BMP8B, a previously unknown secreted factor in cancer progression, has the potential to be used as a prognostic biomarker. The present study may provide insight into a new mechanism that underlies the dissemination of gastric cancer cells.
PMCID: PMC3788827  PMID: 24137334
bone morphogenetic protein 8B; gastric cancer; bone marrow
20.  Inflammation and Oxidative Stress in Diabetic Nephropathy: New Insights on Its Inhibition as New Therapeutic Targets 
Journal of Diabetes Research  2013;2013:248563.
Diabetes and insulin resistance can greatly increase microvascular complications of diabetes including diabetic nephropathy (DN). Hyperglycemic control in diabetes is key to preventing the development and progression of DN. However, it is clinically very difficult to achieve normal glucose control in individual diabetic patients. Many factors are known to contribute to the development of DN. These include diet, age, lifestyle, or obesity. Further, inflammatory- or oxidative-stress-induced basis for DN has been gaining interest. Although anti-inflammatory or antioxidant drugs can show benefits in rodent models of DN, negative evidence from large clinical studies indicates that more effective anti-inflammatory and antioxidant drugs need to be studied to clear this question. In addition, our recent report showed that potential endogenous protective factors could decrease inflammation and oxidative stress, showing great promise for the treatment of DN.
PMCID: PMC3686081  PMID: 23862164
21.  Syringocystadenoma Papilliferum of the Scalp in an Adult Male – A Case Report 
Syringocystadenoma papilliferum is a benign adnexal skin tumour of the apocrine or the eccrine type with characteristic histological features and varied and non-distinct clinical findings. It is relatively a rare neoplasm, which is called as a childhood tumour, since it usually appears at birth or during puberty. A case of syringocystadenoma papilliferum of the scalp in an adult male has been presented, which was clinically diagnosed at first as keratocanthoma of the scalp but was later histologically confirmed as syringocystadenoma papilliferum.
PMCID: PMC3644463  PMID: 23730665
Skin hamartoma; Apocrine sweat glands; Sebaceous nevus; Basal cell carcinoma
22.  Retinal Not Systemic Oxidative and Inflammatory Stress Correlated with VEGF Expression in Rodent Models of Insulin Resistance and Diabetes 
To correlate changes between VEGF expression with systemic and retinal oxidative stress and inflammation in rodent models of obesity induced insulin resistance and diabetes.
Retinal VEGF mRNA and protein levels were assessed by RT-PCR and VEGF ELISA, respectively. Urinary 8-hydroxydeoxyguanosine (8-OHdG), blood levels of C-reactive protein (CRP), malondialdehyde (MDA), and CD11b/c positive cell ratio were used as systemic inflammatory markers. Retinal expression of Nox2, Nox4, and p47phox mRNA levels were measured as oxidative stress markers. TNF-α, inter-cellular adhesion molecule-1 (ICAM-1), IL1β, and activation of nuclear factor κB (NF-κB) were used as retinal inflammatory markers.
Retinal VEGF mRNA and protein expression increased in Zucker diabetic fatty (ZDFfa/fa) rats and streptozotosin (STZ) induced diabetic Sprague-Dawley rats, after two months of disease, but not in Zucker fatty (ZF) rats. Systemic markers of oxidative stress and inflammation were elevated in insulin resistant and diabetic rats. Some oxidative stress and inflammatory markers (TNF-α, IL-6, ICAM-1, and IL1-β) were upregulated in the retina of ZDFfa/fa and STZ diabetic rats after 4 months of disease. In contrast, activation of NF-κB in the retina was observed in high fat fed nondiabetic and diabetic cis-NF-κBEGFP mice, ZF, ZDFfa/fa, and STZ-induced diabetic rats.
Only persistent hyperglycemia and diabetes increased retinal VEGF expression. Some markers of inflammation and oxidative stress were elevated in the retina and systemic circulation of obese and insulin resistant rodents with and without diabetes. Induction of VEGF and its associated retinal pathologies by diabetes requires chronic hyperglycemia and factors in addition to inflammation and oxidative stress.
Only chronic diabetes induced late markers of inflammation and oxidative stress in the retina correlated with increased VEGF expression, but insulin resistance alone caused systemic and retinal inflammation and no increase in VEGF elevation.
PMCID: PMC3753893  PMID: 23197686
23.  A Clinico-Pathological Study on Benign Breast Diseases 
Background: To study the patterns of clinically benign breast disease in females and to co-relate them with the pathological findings.
Methods: One hundred females who attended the Surgery Outpatients Department in Indira Gandhi Medical College and Research Institute, Pondicherry, with various forms of benign breast diseases during the period from October 2011 to September 2012, were studied. Early diagnoses by doing a triple assessment like a clinical examination, FNAC or a core needle biopsy and imaging methods like ultrasonography or mammography, were made within 72 hrs from the first consultation. The clinical diagnoses were compared with the cytological or histological findings wherever possible and their accuracies were evaluated.
Results: Out of the 100 female patients who were studied, 87 patients who presented with breast lumps and fibroadenoma, accounted for 48% of the cases, which was the highest number of patients. Fibrocystic changes and breast abscesses came next with 18% and 12% cases respectively. We detected 3 cases of proliferative disease with atypia and one case with florid hyperplasia, which had high and low risk factors respectively, for developing invasive carcinoma. The oldest lady of the group who was clinically diagnosed to have benign disease, was detected to have invasive ductal carcinoma. They were treated in our hospital and were advised follow up.
Conclusion: Benign breast diseases are common in female patients and fibroadenoma is the commonest of them all. Triple assessment provided a quick diagnosis and it alleviated unnecessary anxiety from the patients about breast cancer. The clinical diagnosis of a breast lump, as confirmed by cytology and histology, was accurate in 91.95 % of the cases.
PMCID: PMC3616566  PMID: 23634406
Benign breast disease; Risk factors; Pathology; Triple assessment
24.  Regulation of the Epithelial Adhesion Molecule CEACAM1 Is Important for Palate Formation 
PLoS ONE  2013;8(4):e61653.
Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at various developmental stages before, during, and after palate fusion using GeneChip® microarrays. Ceacam1 was one of the highly up-regulated genes during palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was present in prefusion palatal epithelium and was degraded during fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1−/−) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1−/− mice. TGFβ3 expression, apoptosis, and cell proliferation in palatal epithelium were not affected in the palate of Ceacam1−/−mice. However, CEACAM1 expression was retained in the remaining MEE of TGFβ-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion.
PMCID: PMC3629100  PMID: 23613893
25.  PKCβ-induced Selective IRS1 Dysfunction and Insulin Resistance in Renal Glomeruli of Rodent Models of Diabetes and Obesity 
Kidney international  2011;79(8):883-896.
Insulin resistance has been associated with the progression of chronic kidney disease in both diabetes and obesity. This study characterizes insulin signaling in renal tubules and glomeruli in insulin resistant and diabetic states.
Insulin-induced phosphorylation of insulin receptor substrate-1 (IRS1), Akt, endothelial nitric oxide (eNOS), and glycogen synthase kinase 3α (GSK3α) were selectively inhibited in the glomeruli but not in the renal tubules of both streptozotocin (STZ)-diabetic and Zucker fatty, insulin resistant rats compared to non-diabetic and Zucker lean rats. Protein levels, but not the mRNA expression, of IRS1 were decreased only in the glomeruli of STZ-diabetic rats and increased its association with ubiquitination. Protein kinase C (PKC) β isoform inhibitor, ruboxistaurin (RBX), treatment enhanced insulin actions and elevated IRS1 expression. In glomerular endothelial cells, high glucose inhibited phosphorylation of Akt, eNOS and GSK3α, decreased IRS1 protein expression and increased association with ubiquitination. Overexpression of IRS1 or the addition of RBX reversed the inhibitory effects of high glucose.
Selective inhibition of the IRS1/PI3K/Akt pathway and insulin activation of eNOS and GSK3α in the glomeruli in diabetes and insulin resistance is partly due to increased IRS1 degradation and PKCβ activation. The loss of insulin's effect on endothelial eNOS and GSK3α activation may contribute to the glomeropathy observed in diabetes and obesity.
PMCID: PMC3612886  PMID: 21228767
diabetic nephropathy; insulin resistance; obesity; insulin receptor substrate-1; protein kinase C β

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